Search

The objective of this study was to ascertain the value of variables measured on bulk tank and composite milk samples as predictors, respectively, of the level of mastitis in herds and individual cows. The standard of comparison was the results obtained by bacteriological examination of the secretion from individual mammary quarters. It was found, whether sampling at the herd or cow level, that of the three variables measured on each sample, namely somatic cell count, percentage of cell volume in channel 8 (includes cells with individual volumes from 89.2 to 178.3 μm3) and presence or absence of Streptococcus agalactiae , that the ability of the first two variables to discriminate (predict) was not significantly improved by the inclusion of the latter variable. Using values for the former two variables as measured on bulk tank milk and collected at the time of quarter sampling, it was possible to correctly classify 45.5% of the study herds into one of the three quarter infection rate categories considered, namely, 25%. The percentage of herds correctly classified was increased to over 80% by inclusion of five monthly counts recorded previous to the time of quarter sampling. Using the same two variables but measured on composite milk samples, 79.4% of cows were correctly classified as being either positive or negative when a positive cow was defined as one having at least one positive quarter. The overall percentage of cows correctly classified decreased from 79.4 to 77.9% when an attempt was made to distinguish between negative cows and cows with one positive quarter. However, this overall percentage progressively increased to 92.7% as the definition of a positive cow was changed from one having one positive quarter to one having two, three or four positive quarters. The procedure shows promise for monitoring the level of mastitis infection in either dairy herds or cows but requires further refinement before consideration could be given to its implementation.

Samples from approximately 1000 bulk tanks from widely separated areas of Ontario were examined over a period of 1 year. Total and differential somatic cell content was estimated electronically. A high degree of reproducibility was shown both in total cell count, and percent of cell volume in specific size ranges (channels) determined with a Coulter Model TA electronic counter. Use of a single count per dilution made little practical difference as compared to using the average of two counts per dilution. There were no significant differences between cell count means and cell volume percentages in channel 8 (cells from 89.2 to 178.3 μm3) from 24- and 48-h-old samples from either fluid or industrial shippers. There was a small significant difference in both measurements in duplicate samples shipped 275 miles before and after formalin fixation, and the cell volume in channel 8 was markedly reduced by shipping samples the same distance. Data from a small group of herds which were quarter sampled and submitted for mastitis laboratory diagnosis indicated the possibility that the percentage of cell volume in channel 8 found in bulk tank milk may be a better indicator of infection incidence in a herd than the total cell count alone.

Electronic counting of somatic cells in farm bulk tank milk, using chemical fat dispersion has been studied. The method used was the Pearson et al. modification of the original Tolle et al. procedure. It was found that fixation of the original sample with formalin was critical, and that with bulk tank milk this was best accomplished by the use of buffered formalin at 30–32 C for 24 h. No significant differences could be found, in samples kept up to 48 h at 4 C, between adding milk to buffered formalin and buffered formalin to milk. Good agreement was found between the DMSCC made on the day samples were taken, and electronic counts obtained following 24 and 48 h refrigeration storage before fixation, and 24 and 48 h following fixation after 24 h refrigeration. Correlation coefficients were + 0.876, + 0.924, and + 0.853, + 0.870, respectively. Replicate counts on both fresh bucket and bulk tank, and single counts on replicate samples from bulk tanks yielded coefficients of variation between 2.98% and 9.69%. The procedure shows promise of providing rapid, accurate somatic cell counts on large numbers of bulk tank milk samples.

SummarySmall numbers of bovine staphylococci, usually less than 30, were infused into the teat cistern of 17 cows secreting less than 100 000 leucocytes/ml milk. Infection was assumed to have occurred if the organisms multiplied sufficiently to be recovered in the foremilk and inflammation was regarded as demonstrated if there was an increase in leucocytes. If the staphylococci were recovered at the 1st post-infusion milking they were consistently recovered thereafter and inflammation always followed but was never evident at the 1st post-infusion milking. Inflammation was usually evident by the 3rd but in one instance was delayed to the 11th post-infusion milking. There was never inflammation in the absence of infection. When there was a high probability that the infusion contained one organism or more, infection occurred in 2 of 10 quarters infused with coagulase-negative staphylococci and 23 of 25 quarters infused with coagulase-positive staphylococci.A significant positive relationship was found between the number of staphylococci found at the 1st post-infusion milking and the number of leucocytes at the 3rd postinfusion milking.

In estimating bacterial populations by the colony count method increased accuracy is obtained by using large numbers of replicates. The high cost of Petri plates makes this difficult in many laboratories. A simple spinner is described which utilizes cheap, easily obtained screw-capped bottles to substitute for Petri plates. The method has proved satisfactory in estimating numbers of Rhizobia, and has resulted in significant savings in the cost of glassware and media, and in incubator space.

SummaryFifty-seven inoculations of a strain of Staphylococcus aureus were made into the outer 4 mm of the teat ducts of 8 cows. The inocula ranged from 10 to 600 colony-forming units (CFU) and were made with a stainless steel instrument designed to overcome the disadvantages of glass rods and cotton swabs hitherto used.A single inoculation resulted in either no colonization of the duct, in temporary colonization for up to 7 days, or in colonization followed by intramammary infection.The recovery of the organisms depended on the size of the inoculum. When 70–100 CFU were placed in the teat duct no organisms were recovered from 12 of 24 quarters after the 1st post-inoculation milking. When 500–600 CFU were used, organisms were recovered from all of 31 quarters for at least 3 milkings, and from 61% for 6 milkings or more, in spite of dipping the teats in a strong disinfectant twice daily.Intramammary infection developed in 1 of 12 quarters (8%) inoculated in the teat duct with about 600 CFU when the animals were milked twice daily, and in 5 of 19 (23%) quarters if the 1st post-inoculation milking was omitted.There was no evidence of sensitization resulting from previous infection.

SummaryA technique for determining the concentration of leucocytes in cow's milk is described which is more rapid and accurate as well as much less tedious than conventional microscope methods. The leucocytes are isolated from the fat globules of comparable size by a novel centrifuging procedure. Sizing of the leucocytes with a Coulter electronic counter shows that their volumes range from about 45 to 1770 µm3but a partially overlapping distribution of other particulate matter prevents the accurate determination of the total number of cells present. The numbers counted in truncated leucocyte distributions, however, correlate linearly with total cell counts determined with an improved microscope method up to 6 × 106cells/ml of milk.

The effects of three changes in the optical equipment used for Direct Microscopic Somatic Cell Count in milk (National Mastitis Council method) on performance of the method have been measured and subjected to statistical analysis of probable significance. Replicate slides of six milk samples were counted by eight technicians among the authors' laboratories. Three levels of cell concentration were represented among the samples. No consistent differences in estimated cell concentration were demonstrated in the comparisons of narrow versus wide reticle band, wide field versus Huyghenian eyepieces, or low (high dry) versus high (oil immersion) magnification. In a few instances within each comparison, individual counters produced markedly different results when the optical equipment was modified. These individual biases were large and consistent with regard to change in objective magnification. No evidence was found to preclude use of wide field eyepieces in performing the DMSCC.

Teats were dipped immediately after milking in (a) an iodophor diluted to contain 1.0 per cent available iodine; (b) P.V.P.—Hibitane, containing 1.6 per cent Hibitane (Chlorhexidine); and (c) P.V.P.—Hibitane containing 0.5 per cent Hibitane. After each cow was milked the teat cups were rinsed briefly and “pasteurized” at 165–170°F for 15 seconds. Teat-dipping alone reduced dramatically the number of micrococci on the inflations but was only partially effective in reducing the percentage of inflations carrying haemolytic staphylococci, except when the 1.6 per cent P.V.P.—Hibitane was used. This material was, however, somewhat irritating to the teats. Teat cup pasteurizing alone partially reduced the percentage of teat cups carrying haemolytic staphylococci, but had little effect on the number of micrococci initially present on the inflations. It appears that the combination of these simple practices will go far towards eliminating the teat-skin and milking machine inflations as reservoirs of haemolytic staphylococci.

ummary Intramammary infusions of crude Beta-hemolysin from Staph. aureus strain 252-F, in amounts of 0.5 MHU or greater, consistently resulted in intense intramammary reaction leading to inflammation. The somatic cell counts in milk of infused glands were significantly elevated by first milking following infusion and usually lasted seven to ten days before dropping to normal levels. The severity of inflammatory reaction and somatic cell responses were roughly proportional to the amounts of Beta-hemolysin infused. Neutrophils obtained from Beta-hemolysin infused mammary glands appeared normal in their physiological properties, which however were significantly inhibited on subsequent incubation with Beta-hemolysin. These observations were confirmed by studies employing partially-purified Beta-hemolysin. Intramammary inoculations of Staph. aureus strain 252-F in small numbers (45 and 90 CFU) resulted in moderate to acute inflammation of the infected glands. Milk from infected glands, as well as those infused with Beta-hemolysin, failed to reveal presence of Beta-hemolysin, on preliminary examination. These studies strongly contradict the view held by most workers, that Beta-hemolytic Staph. aureus and its hemolysin are of little pathogenic importance to the bovine mammary gland. Zusammenfassung Signifikanz von Beta-hamolytischem Staph. aureus als pathogener Keim fur das Rindereuter Euterinfusionen von wenigstens einer halben Haemolysierdose von rohem β-Haemolysin, das von dem Staphylokokkus aureus Stamm 252-F stammte, bewirkte sehr akute Mastitis. Die Anzahl somatischer Zellen in der Milch stieg bedeutend an, was sich schon nach dem ersten Melken zeigte, und meistens sieben bis zehn Tage dauerte, bevor die Zellenanzahl wieder ihren Normalwert erreichte. Die Heftigkeit der Entzundung und der Anstieg der somatischen Zellen war ungefahr proportional zu den verabreichten Mengen des infusierten Haemolysins. Die chemotaktischen und phagozytischen Eigenschaften der Neutrophilen in Eutern, die mit β-Haemolysin infusiert worden waren, waren unverandert. Diese Eigenschaften wurden jedoch erheblich reduziert, sobald die Neutrophilen mit β-Haemolysin bebrutet wurden. Diese Resultate wurden durch Versuche mit teilweise gereinigtem β-Haemolysin bestatigt. Euterinfusionen mit geringen Mengen des Staphylokokkenstamms 252-F ergaben mittelmasige, oder auch akute, Mastitis. Vorlaufige Versuche stellten fest, das weder die Milch von entzundeten Eutern, noch die Milch von β-Haemolysin-injizierten Eutern, β-Haemolysin enthielt. Die vorliegenden Arbeiten widersprechen stark der Ansicht der meisten anderen Autoren, namlich das β-Haemolysine von sehr geringer Bedeutung fur die Pathogenese der Mastitis des Rindes seien. Resume Role de Staph, aureus β-hemolytique en tant que germe pathogene pour la mamelle chez le bovin Des infusions intramammaires d'au moins une demi dose hemolysante de β-hemolysine grossiere a partir d'une souche de Staphylococcus aureus (252-F) provoquerent une mastite tres aigue. Le nombre de cellules somatiques dans le lait augmenta de facon significative deja apres la premiere traite et dura la plupart du temps sept a dix jours avant que le nombre de cellules fut a nouveau normal. L'intensite de l'inflammation et l'augmentation des cellules somatiques fut a peu pres proportionnel a la quantite d'hemolysine administree. Les proprietes chimiotactiques et phagocytantes des neutrophiles infuses avec l'hemolysine β furent inchangees. Ces proprietes furent cependant considerablement reduites du moment ou l'on a incube les neutrophiles avec l'hemolysine β. Ces resultats se confirmerent lors d'essais faits avec une β-hemolysine partiellement purifiee. Des infusions intramammaires faites avec une tres petite quantite de staphylocoques 252-F provoquerent des mastites moyennes mais egalement aigues. Les recherches ont montre que ni le lait des glandes enflammees ni celui des mamelles injectees d'hemolysine β ne contenait d'hemolysine β. Les resultats contredisent fortement le point de vue de la plupart des auteurs qui estiment que l'hemolysine β joue un role mineur dans la pathogenese des mastites chez le bovin. Resumen Significacion del estafilococo dorado betahemolitico como germen patogeno para la glandula mamaria de la vaca Las infusiones mamarias de un minimo de media dosis hemolizante de hemolisina β cruda, procedente de la estirpe Staphylococcus aureus 252-F, ocasionaban una mastitis hiperaguda. Aumentaba considerablemente la cantidad de celulas somaticas en la leche, lo cual ya se evidenciaba despues del primer ordeno, durando casi siempre de siete a diez dias, antes de que la cantidad de celulas volviese a alcanzar su valor normal. La intensidad inflamatoria y el aumento de las celulas somaticas era casi proporcional a las cantidades administradas de hemolisina infundida. Las propiedades quimiotacticas y fagocitarias de los neutrofilos en las mamas que habian sido infundidas con hemolisina β no sufrieron alteracion alguna. Sin embargo, estas propiedades amenguaron bastante tan pronto como se incubaron los neutrofilos con la hemolisina β. Estos resultados se confirmaron mediante ensayos con hemolisina β purificada en parte. Las infusiones mamarias con cantidades escasas de la cepa estafilococica 252-F ocasionaron mamitis medianas, aunque tambien agudas. Los ensayos provisionales establecieron que ni la leche de cuarterones inflamados ni la leche de mamas inyectadas con hemolisina β contenian la hemolisina mencionada. Los trabajos presentes se hallan en contradiccion extrema con el punto de vista de la mayoria de los otros autores, los cuales opinan que las hemolisinas β apenas tienen importancia en la patogenia de la mamitis de vacas.

A new method for quantitation of chemotactic response of polymorphonuclear neutrophils (PMNs) in vitro is described. It involves harvesting PMNs from the chemotactic substance in the lower half of the chamber after a three hour incubation, and expressing their numbers as a percentage of neutrophils initially deposited on the Millipore filters; and is referred to as “Chemotactic index”. In comparison with two techniques commonly used at present, the new method appeared simple, easily reproducible, less time consuming, and more precise in delineating quantitative differences in responses of blood PMNs from different animals to a single chemotactic agent; as well as difference in the potency of two chemotactic agents to PMNs from a single source. The method also enables ready comparison of results from different sources.

The Direct Microscopic Somatic Cell Count can be used advantageously in control of abnormal milk to perform simultaneously screening and confirmation of milk samples. Confidence limits computed according to the individual microscope Strip Factor are used to interpret the count made on a single strip of one of the two milk films. The sample is assigned on the basis of the interpreted count to one of three categories: (a) less than the legal cell concentration maximum ( P

KLECZKOWSKA1 has described a ‘physiological ageing’ of colonies of Rhizobium in an agar layer which renders them immune to attack by bacteriophage after they are 19 hr. old. Thus the multiplication and spread of the phage particles throughout the volume of the plaque must take place within the period set by the ageing of the bacterial colonies.